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Endoplasmic reticulum stress effector CCAAT/enhancer-binding protein homologous protein (CHOP) regulates chronic kidney disease-induced vascular calcification.

Miyazaki-Anzai S, Masuda M, Demos-Davies KM, Keenan AL, Saunders SJ, Masuda R, Jablonski K, Cavasin MA, Kendrick J, Chonchol M, McKinsey TA, Levi M, Miyazaki M - J Am Heart Assoc (2014)

Bottom Line: Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction.This therapy also reduced aortic endoplasmic reticulum stress induced by CKD.These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO (S.M.A., M.M., A.L.K., S.J.S., R.M., K.J., J.K., M.C., M.L., M.M.).

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7‐Ketocholesterol induces mineralization through ER stress response in MOVAS‐1 cells. A, Mineralization and (B) ALP activity of MOVAS‐1 cells treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 7 days. *P<0.05, **P<0.01, and ***P<0.001 vs no treatment. C, Dose‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 24 hours. GAPDH was used as a loading control. D, Time‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with 2.5 μg/mL 7‐ketocholesterol for the indicated time periods (0, 0.5, 3, 6, 12, and 24 hours). E, Mineralization of ATF4‐ or CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 7 days. F and G, ATF4 and CHOP protein expression in ATF4‐ and CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 24 hours. *P<0.05 vs control MOVAS‐1 cells. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; CHOP, C/EBP homology protein; ER, endoplasmic reticulum; MOVAS, mouse vascular smooth‐muscle cell line.
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fig05: 7‐Ketocholesterol induces mineralization through ER stress response in MOVAS‐1 cells. A, Mineralization and (B) ALP activity of MOVAS‐1 cells treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 7 days. *P<0.05, **P<0.01, and ***P<0.001 vs no treatment. C, Dose‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 24 hours. GAPDH was used as a loading control. D, Time‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with 2.5 μg/mL 7‐ketocholesterol for the indicated time periods (0, 0.5, 3, 6, 12, and 24 hours). E, Mineralization of ATF4‐ or CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 7 days. F and G, ATF4 and CHOP protein expression in ATF4‐ and CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 24 hours. *P<0.05 vs control MOVAS‐1 cells. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; CHOP, C/EBP homology protein; ER, endoplasmic reticulum; MOVAS, mouse vascular smooth‐muscle cell line.

Mentions: To examine whether oxysterol‐induced CHOP and ATF4 through ER stress signaling is involved in mineralization and osteogenic differentiation of VSMCs, mouse vascular smooth muscle cell line (MOVAS‐1) cells were treated with 7‐ketocholesterol. CKD significantly increases 7‐ketocholesterol in both humans and mice, as shown in Table 1. 7‐ketocholesterol dose‐dependently increased calcium content and alkaline phosphatase activity (Figure 5A and 5B). ATF4 and CHOP protein levels were significantly increased by 7‐ketocholesterol with dose‐dependent and time‐dependent manners (Figure 5C and 5D). We next treated ATF4 knockdown and CHOP knockdown MOVAS‐1 cells with 7‐ketocholesterol. ATF4 knockdown and CHOP knockdown significantly attenuated oxysterol‐induced mineralization (Figure 5E). ATF4 knockdown decreased ATF4 protein expression induced by 7‐ketocholesterol treatment, resulting in the reduction of oxysterol‐induced CHOP expression (Figure 5F). As expected, CHOP knockdown reduced oxysterol‐induced CHOP expression, but not ATF4 expression (Figure 5G). Treatment with 25‐hydroxycholesterol also induced mineralization and expression of ATF4 and CHOP. Similar to 7‐ketocholesterol, ATF4 and CHOP knockdown both attenuated mineralization and ER stress induced by 25‐hydroxycholesterol treatment (Figure S2).


Endoplasmic reticulum stress effector CCAAT/enhancer-binding protein homologous protein (CHOP) regulates chronic kidney disease-induced vascular calcification.

Miyazaki-Anzai S, Masuda M, Demos-Davies KM, Keenan AL, Saunders SJ, Masuda R, Jablonski K, Cavasin MA, Kendrick J, Chonchol M, McKinsey TA, Levi M, Miyazaki M - J Am Heart Assoc (2014)

7‐Ketocholesterol induces mineralization through ER stress response in MOVAS‐1 cells. A, Mineralization and (B) ALP activity of MOVAS‐1 cells treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 7 days. *P<0.05, **P<0.01, and ***P<0.001 vs no treatment. C, Dose‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 24 hours. GAPDH was used as a loading control. D, Time‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with 2.5 μg/mL 7‐ketocholesterol for the indicated time periods (0, 0.5, 3, 6, 12, and 24 hours). E, Mineralization of ATF4‐ or CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 7 days. F and G, ATF4 and CHOP protein expression in ATF4‐ and CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 24 hours. *P<0.05 vs control MOVAS‐1 cells. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; CHOP, C/EBP homology protein; ER, endoplasmic reticulum; MOVAS, mouse vascular smooth‐muscle cell line.
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Related In: Results  -  Collection

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fig05: 7‐Ketocholesterol induces mineralization through ER stress response in MOVAS‐1 cells. A, Mineralization and (B) ALP activity of MOVAS‐1 cells treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 7 days. *P<0.05, **P<0.01, and ***P<0.001 vs no treatment. C, Dose‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with different concentrations of 7‐ketocholesterol (0, 3, 6, 12.5, and 25 μg/mL) for 24 hours. GAPDH was used as a loading control. D, Time‐dependent effect of 7‐ketocholesterol on ATF4 and CHOP expression. MOVAS‐1 cells were treated with 2.5 μg/mL 7‐ketocholesterol for the indicated time periods (0, 0.5, 3, 6, 12, and 24 hours). E, Mineralization of ATF4‐ or CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 7 days. F and G, ATF4 and CHOP protein expression in ATF4‐ and CHOP‐knockdown MOVAS‐1 cells treated with 7‐ketocholesterol (2.5 μg/mL) for 24 hours. *P<0.05 vs control MOVAS‐1 cells. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; CHOP, C/EBP homology protein; ER, endoplasmic reticulum; MOVAS, mouse vascular smooth‐muscle cell line.
Mentions: To examine whether oxysterol‐induced CHOP and ATF4 through ER stress signaling is involved in mineralization and osteogenic differentiation of VSMCs, mouse vascular smooth muscle cell line (MOVAS‐1) cells were treated with 7‐ketocholesterol. CKD significantly increases 7‐ketocholesterol in both humans and mice, as shown in Table 1. 7‐ketocholesterol dose‐dependently increased calcium content and alkaline phosphatase activity (Figure 5A and 5B). ATF4 and CHOP protein levels were significantly increased by 7‐ketocholesterol with dose‐dependent and time‐dependent manners (Figure 5C and 5D). We next treated ATF4 knockdown and CHOP knockdown MOVAS‐1 cells with 7‐ketocholesterol. ATF4 knockdown and CHOP knockdown significantly attenuated oxysterol‐induced mineralization (Figure 5E). ATF4 knockdown decreased ATF4 protein expression induced by 7‐ketocholesterol treatment, resulting in the reduction of oxysterol‐induced CHOP expression (Figure 5F). As expected, CHOP knockdown reduced oxysterol‐induced CHOP expression, but not ATF4 expression (Figure 5G). Treatment with 25‐hydroxycholesterol also induced mineralization and expression of ATF4 and CHOP. Similar to 7‐ketocholesterol, ATF4 and CHOP knockdown both attenuated mineralization and ER stress induced by 25‐hydroxycholesterol treatment (Figure S2).

Bottom Line: Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction.This therapy also reduced aortic endoplasmic reticulum stress induced by CKD.These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO (S.M.A., M.M., A.L.K., S.J.S., R.M., K.J., J.K., M.C., M.L., M.M.).

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Related in: MedlinePlus